Method of treatment of estrogen deficiency disease

ABSTRACT

2,4-Difluoro-β-estradiol, 2-fluoro-β-estradiol, and related fluoro estrogens used to treat estrogen-deficiency states in human females with genetic predisposition to cancer of estrogen-sensitive tissues.

CROSS-REFERENCE

This application is a continuation-in-part of my application Ser. No.468,519, filed Feb. 22, 1983, now abandoned.

BACKGROUND OF THE INVENTION

Prolonged treatment of experimental animals with exogenous estrogensincluding the steroid hormones--estrone, estradiol and estriol--has beenreported to induce tumors of the testes, endometrium ovary, cervix andlymphoid tissues, mammary tissue, adrenal cortex, kidney, and anteriorpituitary gland. Synthetic estrogens such as DES have also been found toincrease the number of tumors in the same tissues in experimentalanimals.

The exact mechanism of tumor induction by these hormones is unknown. Ithas even been stated that the role of estrogens on the etiology ofbreast cancer and other gynecologic cancers may be permissive ratherthan causative. However, the ultimate question is, "Does theadministration of estrogens under certain conditions, in certain dosagesand for a certain length of time increase the incidence of neoplasms inresponsive tissues?" [Hertz, Cancer, 38, 534 (1976)]. Variousepidemiological studies indicate, without proving, that use of estrogenby post-menopausal women is associated with an increased risk ofendometrial cancer [Goodman & Gilman-The Pharmacological Basis ofTherapeutics, 6th Ed.--pp. 1426, 1429-30 (McMillan, New York 1980)].

It has been proposed, however, that the ring A polyhydroxylatedestrogens, the primary products of estradiol or estrone metabolism,although almost devoid of estrogenic activity themselves, could beresponsible for the carcinogenic or carcinopromoting action ofestradiol. These polyhydroxy metabolites, 2- and 4-hydroxyestradiol or2- and 4-hydroxyestrone, (catechols or ortho-dihydroxybenzenes) are themajor biotransformatory products of estradiol in most species and areknown to bind covalently in vitro to protein or peptides and to DNA.Such binding to protein can be suppressed by prior methylation of thecatechol.

If oxidation of the phenolic ring of an estrogen is indeed a necessarystep in the chain of events leading to increased numbers of tumors inexperimental animals, then interference with metabolic oxidations mightinhibit cancer induction by estrogens. Following this hypothesis, Li andLi, Proc. 72nd Annual Meeting Am. Assoc. Can. Res., 22, 11 (1981), wereable to inhibit diethylstilbestrolinduced renal carcinoma in Syrianhamsters by chronic administration of inhibitors of microsomalcytochrome P-450 enzymes (oxidation enzymes). Another potential approachto prevention of carcinogenesis induction by estrogens would be tomodify the estrogen structure so as to prevent oxidation to a catechol.Krey et al., Catechol Estrogens, Ed-Merriam & Lipsett, pages 249-263,(Raven Press, N.Y. 1983), investigating sexual behavior, such asloridosis, in female rats, found that there is reduced catechol estrogenformation with 2-fluoroestradiol and 4-fluoroestradiol--see FIG. 1, page260. The authors conclude that catechol estrogen formation is notnecessary for lordosis. Catechol estrogens had been shown to beprecursors for reactive intermediates possibly responsible for tissueinjury--Nelson et al., Biochem. Biophys. Res. Comm., 70, 1157 (1976).

The tumorigenicity of dibenzo(a,i)pyrene has been reduced byfluorination at 2,3 and/or 10. It is postulated that oxidation of thehydrocarbon ring to a catechol may be involved in tumor production bythis compound.

There is apparently a genetic predisposition to cancer of estrogendependent tissues in human females. The risk of breast cancer, wherethere is a family history of such tumor in first degree relatives, isincreased 2-3 times [CMA Journal, 121, 505-8 (1979)]. With such persons,the increased risk of cancer potentially induced by administration ofestrogen for treatment of estrogen-deficiency states weighs heavilyagainst such estrogen replacement therapy. Stated in somewhat simplerterms, a human female with a history of greater than normal cancers ofthe breast, cervix, uterus or ovaries in her female relatives will, inmany instances, have to endure the menopause rather than to be able toalleviate hot flashes and other menopausal symptoms by taking anestrogen, all for fear of incurring cancer. It is to such a group ofhuman females, those with a genetic predisposition to cancer of estrogensensitive tissues, that this invention is particularly directed.

It is thus an object of this invention to provide an estrogen ofdecreased cancer induction potential for use in treating menopause orother estrogen deficiency state in human females.

DESCRIPTION OF THE INVENTION

In fulfillment of the above and other objects, this invention provides amethod for alleviating the effects of estrogen-deficiency disease inhuman females having a genetic predisposition to cancer ofestrogen-sensitive tissues comprising the administration of a dose of asteroid according to structure I sufficient to alleviate the symptoms ofsaid estrogen-deficiency disease without increasing the risk in saidhuman female of the development of cancer of estrogen-sensitive tissues.##STR1## wherein R is H or F; when taken singly R¹ is H or ethinyl, andR² is OH; and when taken together, R¹ and R² are O═; and R⁴ is H or CH₃,but can be CH₃ only when R¹ is not H.

Compounds falling within the scope of the above formula include:

2-fluoro-3,17β-estradiol

2,4-difluoro-3,17β-estradiol

2-fluoroestrone

2,4-difluoroestrone

2-fluoro-17α-ethinyl-3,17β-estradiol

2,4-difluoro-17α-ethinyl-3,17β-estradiol

2-fluoro-17α-ethinylestradiol 3-methyl ether

2,4-difluoro-17α-ethinylestradiol 3-methyl ether

While only the 3-methyl ethers of the 17α-ethinyl compounds have beenillustrated above as useful derivatives of the basic fluoroestrogenslisted above, it will be appparent to those skilled in the art thatother derivatives such as the 3-benzoate, 3,17-dipropionate, 3-cypionate(cyclopentylpropionate), 3,17-undecylate, 17-valerate and the like couldbe employed in my novel processes.

Human females falling within the above category in whom risk of cancerof estrogen-sensitive tissues is high are those in whose first degreerelatives--parents, siblings, children--there have developed one or morecancers of estrogen-sensitive tissues.

The most prevalent estrogen-deficiency condition is menopause which canoccur naturally after age 40 or can be induced by destruction of ovarianfunction, either surgically or by irradiation. Since most human femaleswho survive to age 50 will have entered menopause and since menopausalsymptoms are not only unpleasant but may include eventuallyosteoporosis, it is common to treat menopause by making up the estrogendeficiency of menopause by administration of estrogen. Commonlyprescribed estrogens for alleviation of menopausal symptoms include DES(diethylstilbestrol), conjugated equine estrogens, (equilin, estroneetc.), β-estradiol, ethinyl estradiol, mestranol, hexestrol anddienestrol. Obviously, an estrogen with lowered potential for inducingcancer of estrogen sensitive tissues would be invaluable, particularlyfor those human females with a genetic predisposition to such tumors.

Another use of steroid estrogens such as mestranol and ethinyl estradiolhas been as a component of oral contraceptives. Although the estrogendosages for use in the oral contraceptive are less than those used fortreatment of menopausal symptoms, product literature for these estrogenwarns against the use of estrogen-containing oral contraceptives inwomen with known tumors of the breast, uterus, etc. However, there isinsufficient information of a probative nature to determine at this timewhether oral contraceptive users have an increased incidence of cancerof estrogen-dependent tissues. Obviously, it may be advantageous to use2-fluoro-β-estradiol, 2-fluoro-17α-ethinylestradiol or other fluoroestrogens coming within the scope of the above formula, as theestrogenic component of an oral contraceptive, because of the loweredcancer incidence which may be associated with its use.

There is, however, a second property of 2-fluoro-β-estradiol and otherfluoro estrogens represented by I above which may make their use in therelief of estrogen deficiency states or in oral contraceptivesadvantageous. This second property is their resistance to metabolism.Thus, less 2-fluoro-β-estradiol, 2-fluoro-17α-ethinylestradiol or otherfluoro estrogen coming within the scope of formula I may be required toachieve an equal estrogenic effect since circulating estrogen levelswith the fluoro estrogen will remain adequate for a longer period oftime per dose. The fact that less estrogen can be used is probably notas important in reducing cancer incidence as in reducing the incidenceof other diseases such as embolism frequently associated with estrogenadministration in an oral contraceptive. While the quantity ofcirculating estrogen may be the same because of the longer half-life ofthe fluoro estrogen of formula I, the absolute quantity of drug willclearly be less. This lessened incidence of side effects not related toneoplastic incidence or growth is an added advantage of my noveltherapeutic processes.

In order to demonstrate the decreased incidence of estrogen-inducedtumors with the fluoro estrogens of formula I, specifically2-fluoro-β-estradiol, compared to estradiol itself, the followingexperiments were carried out. First, the estrogenicity of the twocompounds plus that of the isomeric 4-fluoro-β-estradiol were measuredby two different methods. In one method, increase in wet uterine weightin ovariectomized, immature rats upon administration of estrogen wasdetermined. The results of this test showed that the fluorinatedestrogens were approximately as estrogenic as estradiol. The followingprotocol was used:

Female Sprague/Dawley rats, 3-4 weeks of age, were ovariectomized. Fourdays after ovariectomy animals were separated into four groups of equalgroup weight (17 animals/group, average weight: 90±1 g/animal) andestrogen treatment was begun. The animals received s.c. injections of3.5 mcg. of estrogen in 0.7 ml. of saline/animal once daily for threedays. Saline solutions were prepared by dissolving the estrogen inethanol and diluting this solution with saline until a 5% ethanol/95%saline mixture was obtained. The control group received only saline. Onthe fourth day, the animals were killed, the uteri were excised, freedof luminal fluid and weighed. Data presented in Table 1 below areaverages of wet uterine weights in mg. and the corresponding standarddeviations.

In a second method, the weights of both testes of those male Syrianhamsters which were used in the in vivo carcinogenicity assay, weremeasured on the 224th day after s.c. implantation of 25 mg. pellets ofestrogen. The weights were measured in grams (expressed as percent oftotal body weight). Treatment of animals with modified estrogens or withestradiol resulted in the shrinking of the testes to approximately 10%of the weights of testes in untreated hamsters.

The following protocol was employed:

Male Syrian hamsters, 3-4 weeks of age (Harlan/Sprague Dawley, Madison,Wis.), were given a 25 mg. s.c. implant consisting of 10% cholesteroland 90% estrogen. A second 25 mg. pellet of the same composition wasimplanted 106 days after the initial estrogen treatment. Estrogentreatment was carried out according to the procedure ofKirkman--National Cancer Institute Monograph, 2, 1-57 (1959). Controlanimals were left untreated. Two hundred twenty-four days aftertreatment with estrogens had begun, hamsters were weighed, killed, bothtestes were excised and their weights measured in grams. Data presentedin Table 1 are the ranges of testes weights from four animals in eachgroup. Weights are expressed as percentages of total body weights.

The results of these two tests are set forth in Table 1. In the table,column 1 gives the name of the compound under test, column 2 the uterinewet weights found and column 3 the weights of testes of male hamsters(as percent of total body weight).

                  TABLE 1                                                         ______________________________________                                        Estrogenic Activity of Estradiols                                                      Range Of Testes Weight                                                                         Uterine Wet Weights                                          Of Male Hamsters Of Female Rats                                      Compound [% Of Total Body Weight]                                                                       [mg] ± s.d.                                      ______________________________________                                        2-fluoro-                                                                              0.18-0.25        92-20                                               β-estradiol                                                              4-fluoro-                                                                              0.18-0.28        118-19                                              estradiol                                                                     estradiol                                                                              0.12-0.26        114-20                                              control  1.85-2.06        33-6                                                ______________________________________                                    

The above estrogenicity measurements compare well with experimentsreported by Heiman et al., J. Med. Chem., 23, 994 (1980) who reportedthe estrogen receptor binding affinities of the fluorinated estrogens.When expressed as ratios of association constants (K_(a) compound/K_(a)estradiol)×100, the binding affinity of 2-fluoro-β-estradiol was foundto be 86, that of 4-fluoro-β-estradiol was 128.

Pfeiffer et al., Proc. Soc. Neuroscience, 8, 52 (1982)--measured theestrogen receptor affinity of 2- and 4-fluoro-β-estradiol in cytosol ofthe hypothalamuspreoptic area, the pituitary, and the uterus. Thefluorinated estradiols were found to have high receptor affinity.Furthermore, biological responses of the fluorinated estrogens, such aselicitation of luteinizing hormone surges, of proceptivity, and oflordosis behavior in rats were, according to Krey et al., (loc. cit.),comparable with those elicited by estradiol.

The carcinogenicity of the modified estrogens was measured in maleSyrian hamsters in vivo. Estrogen, and estrogen alone, causes renalclear cell carcinoma in 100% of the male animal population when exposedto s.c. estrogen implants. This kidney tumor is estrogen-induced andestrogen-dependent for development and growth. Failure to resupplyestrogen every three months or surgical removal of implants results intumor regression within a few weeks. Similarly, a tumor cell line,derived from the Syrian hamster renal clear cell carcinoma was found tobe strongly estrogen dependent in vivo showing a 90% tumor regression 10days after removal of the source of estrogen. The spontaneous kidneytumor incidence in Syrian hamsters without estrogen treatment is verylow. The above facts are fully discussed in Kirkman, Nat'l. Can. Inst.Monograph, 1, 1 (1959); McGregor et al., J. Nat'l. Can. Inst., 24, 1057(1960) and Sirbasku et al., Endocrin., 98, 1260 (1976).

The protocol employed was that set out for determining estrogen activityin male hamsters according to Table 1 (in that the animals examined fortumors at 224 days were the source of the testes). In thecarcinogenicity experiment, further groups of animals were examined at279 and 345 days. The animals were decapitated, the kidneys were excisedand inspected visually for the occurrence of renal celear-cellcarcinoma. Sections were prepared of all kidneys and studiedhistologically. The results of the histologic examinations are set forthin Table 2 below. In the table, column 1 gives the name of the compound,column 2 the total number of animals, column 3, the number of dead orlost animals and columns 4, 5 and 6 results of histologic examination ofanimals at the specified time interval for renal clear-cell carcinoma.

                  TABLE 2                                                         ______________________________________                                        In Vivo Carcinogenicity Of Modified Estrogens                                                   No. of Animals with Tumors/                                                   No. of Animals Examined on                                             No. of Various Days after s.c.                                                Animals                                                                              Implantation of Estrogen                                            No. of   Lost     224    279    345                                   Compound                                                                              Animals  or Dead* Days   Days   Days                                  ______________________________________                                        Estradiol                                                                             18       5        4/4    5/5    0/4                                   2-fluoro-                                                                             15       3        0/4    0/4    0/3                                   β-estradiol                                                              4-fluoro-                                                                             15       2         1/4** 3/6    0/3                                   β-estradiol                                                              Control 10       0        0/3    0/3    0/4                                   ______________________________________                                         *Number of animals lost or dead from various causes during the course of      the experiment. Tumors appear approximately six months after pelleting        with estrogen. Therefore, this group included all hamsters found dead         during the first six months of the experiment, since tumors were not          expected and not evident in the kidneys of these animals. Hamsters found      dead after six months were also included in this group, if their kidneys      could not be recovered. One dead hamster with recoverable kidneys was         included in the nearest examination group at 279 days (animal treated wit     4fluoro-estradiol without renal carcinoma).                                   **2 of 4 judged histologically to be preneoplastic.                      

In Table 2, the fact that animals sacrificed at 345 days afterimplantation were tumor free probably indicates a decrease incirculating estrogen and a regression of the tumors present at 224 daysor at 279 days.

It is apparent from the data in Table 2 that 2-fluoro-β-estradiol has agreatly decreased potential for induced renal clear cell carcinomascompared with estradiol or 4-fluoro-β-estradiol at equal estrogendosages. It is also apparent that merely blocking the ortho oxidation ofestradiol to a catechol as in 4-fluoro-β-estradiol is insufficient byitself to eliminate estrogen-induced renal clear cell carcinomas inhamsters. 2-Fluoro-β-estradiol is thus highly estrogenic yetnon-carcinogenic in the particular animal model. As far as I know, theabove finding is the first to separate estrogenicity and carcinogenicityin the estrogenic hormones.

The fluoroestradiols used in the dose experiments were synthesizedaccording to the procedure of Utne et al., J. Org. Chem., 33, 2469(1968).

An alternate method of preparing 2-fluoro-β-estradiol, in admixture with2-fluoroestrone, is given in French Pat. No. 4878M, Mar. 18, 1965.

An improved method of preparing 2-fluoro-β-estradiol has been developedby Ward and Jones--see Ser. No. 564,595 filed Dec. 22, 1983. Thisprocedure uses 3,17β-estradiol diacetate, a known compound, as astarting material. This compound is mercurated at C-2 as with mercurictrifluoroacetate. The trifluoroacetylmercury group is replaced byfluorine upon treatment with acetylfluoride, after which step thediacetate groups are hydrolyzed to yield 2-fluoro-β-estradiol. A moredetailed description of the Ward-Jones process follows.

About 10 g. of 17β-estradiol-17β-diacetate were dissolved in 15 ml. oftrifluoroacetic acid and the solution cooled to about 0° C. Twelve gramsof mercuric trifluoroacetate were added and the reaction mixture stirredat ice bath temperatures for about 3.5 hours. The solvent was thenremoved by evaporation in vacuo and the residual material dissolved inmethylene dichloride. The methylene dichloride solution was extractedwith water and the water wash discarded. The methylene dichloride layerwas dried with anhydrous sodium sulfate. The drying agent was removed byfiltration and the methylene dichloride evaporated from the filtrate invacuo. The residual foam was triturated with hexane by sonication forabout one hour to produce crystals. The crystallization mixture wasfiltered and the filter cake rinsed with hexane. About 17.2 g. of the2-trifluoroacetylmercury derivative were obtained (92% yield).

Acetyl hypofluorite reagent was prepared as follows.

A suspension of 5 g. of sodium acetate in 50 ml. of glacial acetic acidwas added to 550 ml. of Freon 11 with stirring. The mixture was cooledto about -80° C. in a dry ice-acetone bath while nitrogen was beingbubbled through the reaction mixture. When the reaction mixture hadattained the desired temperature, a stream of 18% fluorine in nitrogenwas bubbled in. After five to six hours, a 2 ml. aliquot was added to 20ml. of 60% aqueous acetic acid containing 1.5 g. of potassium iodide.The iodine generated was completely oxidized by 5.8 ml. of 0.1N sodiumthiosulfate which indicated that the solution was approximately 0.145molar in acetyl hypofluorite.

Two grams of 2-trifluoroacetylmercury-17β-estradiol diacetate weredissolved in 25 ml. of chloroform. To this stirred solution was added 25ml. of the 0.145 molar acetyl hypofluorite mixture prepared as describedabove. After about 10 minutes, the mixture was combined with othersimilar reaction mixtures. The combined reaction mixtures were thenwashed with water, with aqueous saturated sodium bicarbonate and againwith water. The extracts were then dried. Evaporation of the solvent invacuo yielded a residual yellowish oil.

Thirty-eight similar runs employing a total of 76 g. of the2-trifluoroacetylmercury-17β-estradiol diacetate starting material werecombined and the combined residues obtained after workup werechromatographed over silica gel using a 10% ethyl acetate--90% isoctanesolvent mixture as the eluant. Those fractions containing2-fluoro-17β-estradiol diacetate as determined by NMR analysis werecombined to give 9.5 g. of the desired compound. This material wasdissolved in toluene and rechromatographed over a Prep 500 silica gelcolumn with toluene as the eluant. Seven and seven tenths grams of thedesired 2-fluoro-17β-estradiol diacetate were thus obtained.

Three and six tenths grams of 2-fluoro-17β-estradiol diacetate weresuspended in 48 ml. of methanol and 15 ml. of water. A solution of 2.4g. of potassium hydroxide in 29 ml. of water was added. The reactionmixture was stirred overnight at ambient temperature. The volatileconstituents were removed in vacuo and the resulting residue suspendedin 1N aqueous hydrochloric acid. This suspension was slurried with ethylacetate until two clean layers formed. The aqueous layer was separatedand the separated layer washed with additional ethyl acetate. The ethylacetate extracts were combined and the combined extracts washed twicewith water and then dried. The combined solutions were concentrated toless than 50 ml. and crystallization induced by scratching. Thecrystallizing solution was placed at about 0° C. overnight. Crystalsthus produced were separated by filtration and the filter cake washedwith pre-cooled methanol. Two and nine hundredths grams of2-fluoro-17β-estradiol were obtained in this way. The compoundcrystallized with 1/2 mole of methanol.

Analysis for the hemimethanolate of 2-fluoro-17β-estradiol was asfollows:

Calculated: C, 72:51; H, 8.22:

Found: C, 72.30; H, 8.49.

2-Fluoro-3,17β-estradiol can be converted to 2-fluoro estrone and thatcompound in turn converted to 2-fluoro-17β-ethinyl-3,17β-estradiol bystandard procedures useful in preparing 17α-ethinyl-β-estradiol from17β-estradiol and/or estrone--see also French Pat. No. 4878M referred toabove. The corresponding 3-methyl ethers, 17-esters, etc. are alsopreparable by standard procedures used in preparation of thecorresponding non-fluorinated derivative.

For use in treatment of estrogen deficiency states such as the menopausecompounds coming within the scope of formula I, such as2-fluoro-β-estradiol and 2-fluoro-17α-ethinyl estradiol, can beformulated for oral administration in the form of tablets containing 1-2mg. of drug per tablet. Preferably, the drug is micronized beforeincorporation into tablets and care is taken in tabletting not to fusethe microscopic particles into larger aggregates. Another useful solidformulation would be a buccal tablet incorporating 2-fluoro-β-estradiolas the estrogenic drug. A commonly used method of administeringβ-estradiol itself is by implanting a 25 mg. pellet subcutaneously. Thepellet pays out less than 2 mg./day of the drug. 2-Fluoro-β-estradiolcan be similarly pelleted and implanted. An intramuscular suspension of2-fluoro-β-estradiol in sterile, isotonic saline is also a satisfactoryformulation. If a depot effect is desired, a suspension in a vegetableoil such as sesame oil should be used. The daily dose of compoundscoming within the scope of formula I such as 2-fluoro-17α-ethinylestradiol, 2,4-difluoro-β-estradiol, 2,4-difluoro-17α-ethinyl estradioland 2-fluoro-β-estradiol etc. is 1-2 mg. but should not exceed the upperlimit. If pellets or depot formulations are employed, the estrogenic payout (amount released to the female body) should be 2 mg. or less perday.

Esters of 2-fluoro-β-estradiol can also be employed in IM suspensions togive an added depot effect, such esters including for example the3-benzoate, the 3,17-dipropionate and the 17-cyclopentylpropionate. Withthese IM formulations, the rate of release should not be greater than 2mg./day of 2-fluoro-β-estradiol equivalent. Thus, where esters are used,the highest daily dose of ester should be no higher than a doseequivalent to 2 mg. of 2-fluoro-17-β-estradiol.

The 3-methyl ethers of 2-fluoro-17α-ethinyl estradiol and2,4-difluoro-17α-ethinyl estradiol are particularly useful as theestrogenic component of a typical oral contraceptive regimen, and areused at dosage rates 100-200% of that of mestranol, the correspondingunfluorinated compound.

I claim:
 1. The method of alleviating menopausal symptoms in humanfemales having an inherited susceptibility to cancer of estrogensensitive tissues which comprises administering to said menopausalfemale a menopausal symptom relieving dose of a compound of the formula##STR2## wherein R is H or F; R¹, when taken singly, is H or ethinyl;R², when taken singly, is OH; R¹ and R², when taken together, are O═ andR⁴ is H or CH₃, but can be CH₃ only when R¹ is not H, not to exceed 2mg./day, thereby reducing the probability of said human femaledeveloping cancer in estrogen sensitive tissues induced by the ingestionof estrogens to relieve menopausal symptoms.
 2. In the treatment ofmenopause or other estrogen deficiency disease in human females having ahigh genetic predisposition for cancer of estrogen sensitive tissues,the improvement which comprises the administration to such a female ofan amount of a compound of the formula ##STR3## wherein R is H or F; R¹when taken singly, is H or ethinyl; R², when taken singly is OH; R¹ andR² when taken together, are O═ and R⁴ is H or CH₃ but can be CH₃ onlywhen R¹ is not H, sufficient to relieve symptoms of estrogen deficiency,but not to exceed 2 mg./day, while simultaneously reducing the chance ofdevelopment of cancer of estrogen sensitive tissues ordinarilydeveloping on estrogen treatment in such females.
 3. A process fortreating estrogen deficiency disease in human females without increasingthe incidence of neoplasms of estrogen-sensitive tissues which comprisesadministering an estrogenic dose of a compound of the formula ##STR4##wherein R is H or F; R¹, when taken singly, is H or ethinyl; R², whentaken singly is OH; R¹ and R² when taken together, are O═ and R⁴ is H orCH₃ but can be CH₃ only when R¹ is not H, sufficient to overcome saiddeficiency to such human female, but not to exceed 2 mg./day, on a dailybasis.
 4. A method for alleviating the effects of estrogen-deficiencydisease in human females having a genetic predisposition to cancer ofthe estrogen sensitive tissues comprising the administration of a doseof a compound of the formula ##STR5## wherein R is H or F; R¹, whentaken singly, is H or ethinyl; R², when taken singly is OH; R¹ and R²,when taken together, are O═ and R⁴ is H or CH₃ but can be CH₃ only whenR¹ is not H, sufficient to alleviate the symptoms of saidestrogen-deficiency disease without increasing the risk in said humanfemale of the development of cancer of estrogen-sensitive tissues, saiddose not to exceed 2 mg./day.
 5. A process according to claim 1 in whichthe fluoro estrogen administered is 2-fluoro-β-estradiol.
 6. A processaccording to claim 2 in which the fluoro estrogen administered is2-fluoro-β-estradiol.
 7. A process according to claim 3 in which thefluoro estrogen administered is 2-fluoro-β-estradiol.
 8. A processaccording to claim 4 in which the fluoro estrogen administered is2-fluoro-β-estradiol.
 9. A process according to claim 1 in which thefluoro estrogen administered is 2-fluoro-17α-ethinyl estradiol.
 10. Aprocess according to claim 2 in which the fluoro estrogen administeredis 2-fluoro-17α-ethinyl estradiol.
 11. A process according to claim 3 inwhich the fluoro estrogen administered is 2-fluoro-17α-ethinylestradiol.
 12. A process according to claim 4 in which the fluoroestrogen administered is 2-fluoro-17α-ethinyl estradiol.